We propose to establish the Pittsburgh Center for HIV Protein Interactions (PCHPI), an interdisciplinary center that will specialize in developing methodologies and tools for deriving spatial atomic and cellular level structures of protein-interaction networks and for following the temporal sequence of key events after virus- cell fusion and before HIV genome integration (immediate, post-entry events). The PCHPI will bring scientists and facilities of the highest caliber together for elucidating the interactions of HIV proteins with host cell factors and will provide methodologies and tools to the HIV community at large. By accomplishing the proposed aims, the PCHPI will provide insight into an important area of HIV pathogenesis and open doors for exploring and developing alternative anti-HIV strategies. The center will integrate cellular imaging, biochemistry, and structural biology approaches and aims to: 1) Engage virologists, cell biologists, and structural biologists in a collaborative effort toward understanding HIV-host protein interactions and their structures;2) Develop a framework for computationally predicting functionally important cellular interaction partners for HIV proteins and for experimentally validating in cells, that identified HIV-cellular protein interactions are relevant to HIV pathogenesis;3) Establish a protein production core for HIV-host protein complexes using bacterial and eukaryotic expression systems;4) Establish NMR and X-ray crystallography screening and structure determination cores for HIV accessory/regulatory proteins and their host protein complexes;and 5) Develop a robust HIV and cellular imaging program that combines cryo-electron tomography with cellular and single particle imaging methodologies. Results provided by the proposed research are expected to have major implications in the global fight against AIDS, still considered an incurable disease that afflicts 40 million individuals to date, and new therapeutic strategies and novel drug targets to combat the spread of infection is still a pressing need. Identifying and characterizing atomic structures of key interactions and pivotal events in the immediate post entry stage of the viral lifecycle will open new avenues in this endeavor.